Quinoxalinemethanol compounds for combatting swine dysentery and as growth promoting factors, method of preparation, and compositions containing them

ABSTRACT

The invention relates to new compounds useful for combatting swine dysentery, endowed with growth promoting action but devoid of mutagenic action. 
     Furthermore, the invention relates to the method for the preparation of the compounds of the invention and to compositions containing them.

This invention relates to compounds useful for combatting swine dysentery, endowed with growth promoting action, but devoid of mutagenic action.

Another object of the present invention is the method for the preparation of the compounds of the invention as well as the compositions containing them.

Swine dysentery, also known as vibrionic dysentery or hemorragic dysentery, is an enteric disease primarily characterized by muco-hemorragic diarrhear with large intestine lesions. It is known that Treponema hyodysenteriae is the etiologic agent involved with the swine dysentery. Hitherto, swine dysentery was combatted by constant feeding of antibacterial agents with therapy based on the use of high doses of the above mentioned drugs. However, drugs hitherto used, even when administered at abnormally high doses did not give the desired results.

U.S. Pat. No. 4,086,345 discloses and claims quinoxaline derivatives useful for treating swine dysentery and U.S. Pat. No. 4,128,642 discloses and claims the above compounds as growth rate increasing factors. However, the compounds disclosed in the above mentioned patents are endowed with mutagenic action. Therefore, the use of the above mentioned quinoxaline derivatives is prohibited in some countries, whereas they are tolerated in other countries. As known as men eat the meat of animals treated with the above mentioned quinoxaline derivatives, there is an undoubted risk for animals and men, because of the mutagenic action of those compounds.

Another risk exists for ecology in that waste materials from breedings contain mutagenic compounds.

Therefore, there was a continuing need for drugs of low toxicity and high potence against Treponema hyodysenteriae, but devoid of mutagenic action when tested by the conventional Ames test (see the well known "Handbook of Mutagenicity Test Procedures", Ed. Kibey et al, Elsevier/North Holland Biochemical Press, 1977).

It has been now found that the compounds of the general formula (I): ##STR1## (wherein R is H or a 1-4 C alkyl group) are efficacious against Treponema hyodysenteriae, have a growth promoting action, but are wholly devoid of mutagenic action.

In particular, the compound of formula (I) wherein R=H, the quinoxalyl-N¹,N⁴ -dioxide-2-hydroxy-5-methoxy-phenylcarbinol, is of a low order toxicity and shows a clearly specific action against Treponema hyodysenteriae. Such a specific action is very important in that allows the prophylatic administration of this compound, in that it does not change the intestinal flora. Furthermore, it is an effective growth promoting factor. Quinoxalyl-N¹,N⁴ -dioxide-2-hydroxy-5-methoxy-phenylcarbinol will be indicated hereafter as G 8 compound.

Another object of the present invention is the method for the preparation of the compounds comprised in the general formula (I), by reacting 2-quinoxaline-carboxyaldehyde-N¹,N⁴ -dioxide with 4-methoxyphenolmagnesium chloride, optionally substituted. The reaction occurs in an apolar solvent, at room temperature, the molar ratio of the two reagents being 1:1.

The compounds of this invention can be administered by oral route incorporated in the feed ration. They can be intimately mixed with a generally used swine feed ration, to prepared a homogeneous feed ration. The term "feed ration" in this invention is intended to mean the food for the swine and it is not intended that the invention is limited thereby. Preferably, a compound according to the present invention is thoroughly mixed with the feed ration so that it is uniformly dispersed throughout. It is also possible to sprinkle it on the daily food supplies in the form of a powder or as pellets.

The amount of the compound to be added to swine feed rations is generally comprised between 25 and 500 g/ton.

Compound G 8 has been tested "in vitro" against Treponema hyodysenteriae by a known technique. The minimum inhibitory concentration (the lowest concentration of compound in a dilution series where growth is inhibited) was 0.1 μg/ml.

The minimum bacteriocidal concentration (the lowest concentration of compound in which no viable Treponemes are observed upon dilution and subculture from the broth onto blood agar plates) was greater than 0.1 μg/ml but less than 1 μg/ml.

Furthermore, G8 compound was tested for acute toxicity by several modes of administration in four species, namely mouse, rat, guinea pig and rabbit. The compound was found to be of low order of toxicity. The test results are given below in tables 1, 2, 3 and 4.

                  TABLE 1                                                          ______________________________________                                                  Acute toxicity of G 8 in female mice                                  Dosage   Dead/Treated animals after                                            mg/kg    1 day   2 days     4 days 7 days                                      ______________________________________                                                Endoperitoneal administration                                           2000     6/6                       6/6                                         1000             6/6               6/6                                          500                               6/12                                         250                               0/18                                               Oral administration                                                     .sup.   0.sup.x                  0/6                                           4000                    1/12     1/12                                          2000                             0/12                                          1000                             0/12                                          ______________________________________                                          .sup.x only the vehicle                                                  

                                      TABLE 2                                      __________________________________________________________________________      Acute Toxicity of G 8 in the Rat                                              __________________________________________________________________________     a. First Experiment                                                               Route of Ad-                                                                               Dead/Treated                                                                           Body Weight                                                                           (m ± SEM)                                                                           Statistical                              Sex                                                                               ministration                                                                           mg/kg                                                                              within 21 days                                                                         in g. Start                                                                           Termination                                                                            Significance*                            __________________________________________________________________________     M  oral    4000                                                                               0/4      234.5 ± 13.8                                                                      288.7 ± 13.8                                                                        t 0.05                                   M  oral    .sup.   0.sup.x                                                                    1/4     233.7 ± 3.7                                                                        331.0 ± 0.5                                   F  oral    4000                                                                               0/4     201.2 ± 4.2                                                                        238.2 ± 12.1                                                                        t 0.05                                   F  oral    .sup.   0.sup.x                                                                    1/4     189.2 ± 3.9                                                                        230.0 ± 10.5                                  M  endoperitoneal                                                                          500                                                                               1/4     234.0 ± 6.2                                                                        314.3 ± 10.3                                                                        t 0.05                                   M  endoperitoneal                                                                         .sup.   0.sup.x                                                                    0/4     230.0 ± 5.7                                                                        324.0 ± 8.7                                   F  endoperitoneal                                                                          500                                                                               2/4     206.2 ± 8.7                                                                         286.0 - 272.0                                                                         t 0.05                                   F  endoperitoneal                                                                         .sup.   0.sup.x                                                                    0/4     207.5 ± 4.3                                                                        253.5 ± 7.7                                   __________________________________________________________________________     b. Second experiment                                                              Route of      Dead/Treated                                                                             Body weight                                                                             (±SE)                                   Sex                                                                               Administration                                                                         mg/kg within 7 days                                                                            in g. Start                                                                             Termination                                __________________________________________________________________________     M  oral    4000  0/4       222.5 ±  6.2                                                                         231.7 ± 15.7                            F  oral    4000  0/4        252.0 ± 16.6                                                                        253.5 ± 12.1                            M  intraperitoneal                                                                         500  2/4       226.2 ± 6.8                                                                          225.0 - 212                                F  intraperitoneal                                                                         500  0/4       232.5 ± 5.9                                                                          218.2 ± 7.0                             __________________________________________________________________________     c. Cumulative data regardless of animal sex                                    Route of administration                                                                         mg/kg    Dead/Treated within 7 days                           __________________________________________________________________________     Oral             .sup.   0.sup.x                                                                         0/8                                                  oral             4000     0/16                                                 intraperitoneal  .sup.   0.sup.x                                                                         0/8                                                  intraperitoneal   500     4/16                                                 __________________________________________________________________________      .sup.x Only the vehicle was administered by the same route                     *Student's t test                                                        

                  TABLE 3                                                          ______________________________________                                         Acute toxicity of G 8 in the guinea pig by oral administration                 Dosage                                                                         mg/kg     Dead/Treated within 21 days                                          ______________________________________                                          500      0/4                                                                  1000      1/4                                                                  2000      5/6                                                                  4000      6/6                                                                  0.sup.x    0/13                                                                ______________________________________                                          .sup.x Only the vehicle was administered                                 

                  TABLE 4                                                          ______________________________________                                         Acute toxicity of G 8 in the rabbit by esophageal administration               Dosage Dead/Treated Body weight (m ± SE)                                    mg/kg  within 7 days                                                                               in g. Start Termination                                    ______________________________________                                         2000   0/2          2250-2150   2180 + 2140                                    1000   0/4           2037 ± 104.3                                                                           1922.5 ± 71.5                               0.sup.x                                                                               0/4          2135 ± 75                                                                               2262 ± 215                                   500   0/2          2000-2100   1650-1550                                      ______________________________________                                          .sup.x Only the vehicle was administered                                 

In view of the favorable acute toxicity data, compound G 8 was administered orally in sub-acute, but relatively large doses, to mice and rats for 15 days. Results are given in Tables 5 and 6.

                                      TABLE 5                                      __________________________________________________________________________     Subacute toxicity of G 8 in the mouse                                          Daily dose: 500 mg. G 8 by gastric gavage for 15 days                                            % Body weight change                                                                       Fresh organ to body weight ratio                 Oral treatment                                                                           Dead/Treated                                                                           (m ± SE) Liver   Kidneys                                  __________________________________________________________________________     Vehicle   0/10    20.4 ± 4.2                                                                              5.2 ± 0.2                                                                           1.4 ± 0.1                             G 8, 500 mg/kg/day                                                                       0/10    -8.1 ± 3.9                                                                              5.9 ± 0.3                                                                           1.5 ± 0.1                             __________________________________________________________________________     a. Mortality and body weight                                                   Daily dose: 1 g/kg/day for 15 days                                             Oral treatment      Dead/Treated                                                                              % Body weight change                            __________________________________________________________________________     Vehicle (H.sub.2 O) 0/12       24.54 ± 0.64                                 G 8 in water, 1 g/kg/day                                                                           2/12        18.5 ± 0.75                                 Vehicle (adraganth gum) (x)                                                                        0/12       25.04 ± 1.18                                 G 8 in adraganth gum                                                                               3/12       16.27 ± 1.31                                 __________________________________________________________________________     b. SGOT and SGPT (24 hrs. after last dose)                                                         Units/ml                                                   Oral treatment      SGOT         SGPT                                          __________________________________________________________________________     Vehicle:                                                                       Water               116          4                                             Adraganth gum       119          6                                             G 8 in water        124          9                                             G 8 in adraganth gum                                                                               132          10                                            __________________________________________________________________________

                  TABLE 6                                                          ______________________________________                                         Subacute toxicity of G 8 in female rats                                        Daily dose: 22 g/kg/day of G 8 in female rats by gastric                       gavage for 21 days                                                                      Dead/   Body weight in g (m ± SE)                                  Oral treatment                                                                            Treated   Start       Termination                                   ______________________________________                                         Vehicle    2/6.sup.x 200.0 ± 4.1                                                                             233.2 ± 5.1                                G 8, 2 g/kg/day                                                                           1/6.sup.x 204.1 ± 2.0                                                                             210.6 ± 9.6                                ______________________________________                                         Daily dose: 2 g/kg/day of G 8 by gastric gavage for 21 days                    Oral    Average percent weight of fresh organs (m ± SE)                     Treatment                                                                              Lung          Liver      Kidneys                                       ______________________________________                                         Vehicle 0.85 ± 0.06                                                                               3.45 ± 0.07                                                                            0.95 ± 0.04                                (3 animals)                                                                    G 8     1.08 ± 0.09 NS                                                                            4.54 ± 0.10                                                                            1.04 ± 0.3 NS                              (5 animals)                                                                    ______________________________________                                          .sup.x Death caused by a mistake in esophagus incannalulation. Diagnosis       was confirmed at the postmortem examination                              

Chronic toxicity has been studied in female mice. Results were very satisfactory.

Furthermore, teratogenetic study was conducted with male and female mice and rats. The number of young delivered live at birth was comparable with controls. No malformations in either group were observed.

The compositions containing swine feed ration and as active ingredient, an effective dose of a compound comprosed in the general formula (I) are a further object of the present invention.

The compositions according to the present invention may be in form of powders or pellets.

The following examples are given to illustrate the present invention, without limiting it in any way.

EXAMPLE 1

A solution of 400 ml (1 mol) of methylmagnesium chloride in 600 ml of tetrahydrofuran is added dropwise with 124 g (1 mol) of 4-methoxyphenyl in 100 ml of tetrahydrofuran, under nitrogen and while keeping the temperature under 15° C.

2000 ml of benzene are then added and the solution is then concentrated, under a 15-20 mm vacuum, to about 2200 ml.

190 g (1 mol) of 2-quinoxaline-carboxyaldehyde-N¹,N⁴ -dioxide in 1000 ml of benzene are then added to the above concentrated solution and the mixture is kept under stirring overnight, at room temperature.

After having neutralized with 10% hydrochloric acid, the aqueous phase is discarded and the organic phase is evaporated under a 15-20 mm vacuum. By crystallization from acetone and decoloration with activated coal, there are obtained 156 g of the desired product. Yield 50% on the theoric. m.p. 138°-140° (dec.).

The elemental analysis gave the following results: (%)

    ______________________________________                                                    Calculated                                                                             Found                                                       ______________________________________                                         C            61.34     61.25                                                   H            4.18      4.21                                                    N            8.34      8.36                                                    ______________________________________                                    

IR and NMR spectra confirm the foreseen structure.

EXAMPLE 2

Compound G 8 is mixed with swine feed rations at a level of 200 g/ton and was fed to swine housed in an area where there has been a previous outbreak of swine dysentery. Another herd was housed in a similar area, where there has been a previous outbreak of dysentery. They were fed the same rations as the first herd, but with no G 8 or other drug.

Many members of the herd developed symptoms of dysentery. G 8 was then added with the rations at a level of 400 g/ton. The spread of the disease was halted and the diseased members became free from dysentery symptoms.

EXAMPLE 3

The effect of compound G 8 was tested in feed rations for baby-swines.

Compound G 8 was added at a dosage level of 100 g/ton of feed. Five replicate groups of 10 animals (5 males and 5 females), age one day, were fed the ration containing G 8 and compared to 5 replicate groups of animals (5 males and 5 females) which received the same ration but without G 8.

The test period was 28 days. The results obtained are given in the following table:

    ______________________________________                                                       Body weight gain/swine                                                         Ration without Ration with                                                     G 8            G 8                                               ______________________________________                                         Males           6150     g       6480 g                                        Females         5740     g       5880 g                                        Combined average                                                                               6000             6190                                          Improvement, %  --               3.1                                           Feed/gain ratio 1.82             1.77                                          Improvement, %  --               2.8                                           ______________________________________                                     

I claim:
 1. A compound for combatting swine dysentery, endowed with growth promoting action, but devoid of mutagenic action when tested by the conventional Ames test, and having the formula (I): ##STR2## wherein R is H or a 1-4 C alkyl group.
 2. Alpha(2-hydroxy-5-methoxy phenyl)quinoxaline-2-methanol, N¹ N⁴ dioxide.
 3. A swine feed composition comprising a nutritional swine feed and as active ingredient a compound of claim 1, in an amount between 25 and 500 g/ton.
 4. A growth promoting factor comprising as active ingredient a compound of the formula (I) of claim 1, in association with a pharmaceutically acceptable carrier. 